The Melanoma Inhibitor of Apoptosis Protein: A Target for Spontaneous Cytotoxic T Cell Responses  Mads Hald Andersen, Sine Reker, Jürgen C. Becker, Per thor Straten  Journal of Investigative Dermatology  Volume 122, Issue 2, Pages 392-399 (February 2004) DOI: 10.1046/j.0022-202X.2004.22242.x Copyright © 2004 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Identification of HLA-A2-binding peptides from ML-IAP. Class I MHC heavy chain bands were quantified on a PhosphorImager. The mount of stabilized HLA-A2 heavy chain is directly related to the binding affinity of the added peptide. The binding of the HLA-A2-restricted-positive control peptide HIV Pol476 (black square) was compared with the peptides ML-IAP90 (black triangle), ML-IAP230 (white square), ML-IAP245 (black circle), and ML-IAP280 (white circle). Journal of Investigative Dermatology 2004 122, 392-399DOI: (10.1046/j.0022-202X.2004.22242.x) Copyright © 2004 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 T cell response against the ML-IAP280 (QLCPICRAPV) peptide. (A) Strong responses was seen in TIL from the melanoma patients Cmel72 and Cmel9 and in PBL from the melanoma patient Cmel3. T lymphocytes were stimulated once with peptide before plated at 3×105 cells per well in duplicates either with T2 cells without (light gray) or with peptide (dark gray). The graph depicts the quantification of interferon-γ producing cells. (B) Patients hosting weaker ML-IAP280 responses. The average number of ML-IAP280-specific cells was calculated after subtraction of spots without added peptide for each patient using the ImmunoSpot Series 2.0 Analyzer (CTL Analyzers, LLC). PBL from 45 melanoma patients and TIL from 16 patients was examined. p<0.05 (evaluated by Student's t test for unpaired samples) calculated as the number of spots in wells with added peptide as compared with the number of spots in wells without peptide. Journal of Investigative Dermatology 2004 122, 392-399DOI: (10.1046/j.0022-202X.2004.22242.x) Copyright © 2004 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 T cell response against the peptides ML-IAP245 (RLQEERTCKV), ML-IAP230 (VLEPPGARDV), and ML-IAP90 (RLASFYDWPL). TIL samples from 20 patients and in PBL from 37 melanoma patients were analyzed. T lymphocytes were stimulated once with peptide before plated at 3×105 cells per well in duplicates either without or with peptide. The average number of peptide-specific spots (after subtraction of spots without added peptide) was calculated for each patient using the ImmunoSpot Series 2.0 Analyzer (CTL Analyzers, LLC). p<0.05 (evaluated by Student's t test for unpaired samples) calculated as the number of spots in wells with added peptide as compared with the number of spots in wells without peptide. Journal of Investigative Dermatology 2004 122, 392-399DOI: (10.1046/j.0022-202X.2004.22242.x) Copyright © 2004 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 T cell response against the peptide ML-IAP280 (QLCPICRAPV) in healthy individuals. PBL from 34 HLA-A2-positive, healthy individuals were analyzed. T lymphocytes were stimulated once with peptide before plated at 3×105 cells per well in duplicates either without or with peptide. The average number of peptide-specific spots (after subtraction of spots without added peptide) was calculated for each patient using the ImmunoSpot Series 2.0 Analyzer (CTL Analyzers, LLC). p<0.05 (evaluated by Student's t test for unpaired samples) calculated as the number of spots in wells with added peptide as compared with the number of spots in wells without peptide. Journal of Investigative Dermatology 2004 122, 392-399DOI: (10.1046/j.0022-202X.2004.22242.x) Copyright © 2004 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 In situ detection of ML-IAP-reactive CTL. Confocal laser scanning microscopy was used to detect CTL reacting with a Cy3-conjugated anti-CD8 antibody (red channel) and a fluorescein isothiocyanate-conjugated multimeric HLA-A2/ML-IAP280 construct (green channel) (first and second columns) or with a fluorescein isothiocyanate conjugated multimeric HLA-A2/ML-IAP245 construct (last column) in primary tumors from two HLA-A2-positive melanoma patients. Journal of Investigative Dermatology 2004 122, 392-399DOI: (10.1046/j.0022-202X.2004.22242.x) Copyright © 2004 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 Cytolytic capacity of ML-IAP-specific CTL. (A) ML-IAP245-reactive CTL were isolated from PBL from the melanoma patients CmelE22 using peptide-coated magnetic beads before being plated at 2×103 cells per well in duplicates either with T2 cells without or pulsed with ML-IAP245. (B) ML-IAP280-reactive CTL were isolated from a melanoma-infiltrated lymph node from patient Cmel72 using peptide-coated magnetic beads. These cells was analyzed for specific lysis of T2 cells with (square) or without (triangle) ML-IAP280 peptide. (C) Lysis by ML-IAP280-isolated T cells of the autologous melanoma cell line FM72 (square), the HLA-A2-positive melanoma cell line FM93 (triangle), the HLA-A2 negative cell line FM56 (black circle) and the natural killer target cell line K562 (white circle). Journal of Investigative Dermatology 2004 122, 392-399DOI: (10.1046/j.0022-202X.2004.22242.x) Copyright © 2004 The Society for Investigative Dermatology, Inc Terms and Conditions